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In-vivo energy depleting strategies for killing drug-resistant cancer cells |
| 7514413 |
In-vivo energy depleting strategies for killing drug-resistant cancer cells
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| Patent Drawings: | |
| Inventor: |
Martin, et al. |
| Date Issued: |
April 7, 2009 |
| Application: |
10/518,003 |
| Filed: |
June 13, 2003 |
| Inventors: |
Martin; Daniel S. (Pound Ridge, NY)
Bertino; Joseph R. (Branford, CT)
Koutcher; Jason (New Rochelle, NY)
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| Assignee: |
Sloan-Kettering Institute for Cancer Research (New York, NY) |
| Primary Examiner: |
Jiang; Shaojia Anna |
| Assistant Examiner: |
Crane; Lawrence E. |
| Attorney Or Agent: |
Law Offices of Albert Wai-Kit Chan, PLLC |
| U.S. Class: |
514/34; 514/269; 514/45; 514/553; 514/75 |
| Field Of Search: |
514/45; 514/269; 536/27.21; 546/310; 546/348; 562/8; 562/11; 562/571; 562/533; 564/47; 568/926 |
| International Class: |
A61K 31/70; A61K 31/185; A61K 31/505; A61K 31/66 |
| U.S Patent Documents: |
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| Foreign Patent Documents: |
WO93/23014; WO 93/23014; PCT/US01/46886; WO 02/45720; PCT/US03/18716 |
| Other References: |
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| Abstract: |
This invention also provides a method for treating a cancer subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor or an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, wherein said composition produces a substantially better effect than a composition without at least one of the ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor and an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside. |
| Claim: |
What is claimed is:
1. A pharmaceutical composition comprising an effective amount of N-(phosphonacetyl)-L-aspartic acid (PALA), alanosine (AL), 6-methylmercaptopurine riboside (MMPR), and3-bromopyruvate (BrPA), in combination with a pharmaceutically acceptable carrier.
2. The composition of claim 1, further comprising adriamycin.
3. The composition of claim 2, wherein the amount of adriamycin is one-half the maximum tolerated dosage.
4. A method for treating a subject having breast cancer, comprising administering to the subject the following agents: (i) N-(phosphonacetyl)-L-aspartic acid (PALA); (ii) alanosine (AL); (iii) 6-methylmercaptopurine riboside (MMPR); and (iv)3-bromopyruvate (BrPA).
5. The method of. claim 4, wherein the agents are administered concurrently or sequentially.
6. The method of 4 further comprising administration of adriamycin.
7. The method of 6, wherein the amount of adriamycin administered is one-half the maximum tolerated dosage. |
| Description: |
Throughout this application, various references are referred to. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION AND CANCER RELEVANCE
Drug resistance is the central problem of cancer chemotherapy. Clinically effective combination chemotherapy can cause impressive objective tumor response rates, including complete tumor regressions, but some cancer cells are of lessersensitivity to the agent (i.e., are drug-resistant), are only damaged, recover, and re-grow. The delayed tumor recurrence yields only a short remission period with little improvement in survival time.
There are many mechanisms of drug resistance. Multiple independent mechanisms of drug resistance may coexist in a population of tumor cells as well as in the same cancer cells, as they arise from multiple genetic changes in single cell clones,and are part of the heterogeneity of the neoplastic process. Mechanisms of drug resistance have been largely identified, and this knowledge has suggested many specific approaches to overcoming one or another type of clinical drug resistance, but theseattempts have failed as they also potentiate drug toxicity towards normal tissues.
The therapeutic research strategy for several decades has been that the administration of multiple drugs with different properties and mechanisms of action at optimal doses and intervals should result in cells resistant to one class of drug beingkilled by another drug in the regimen. However, the extensive, clinical data over these decades has evidenced only a minor impact on treatment outcome along with troublesome and serious toxic side effects (e.g., emesis, diarrhea, alopecia, asthenia,fatigue, myelosuppression, febrile neutropenia requiring hospitalization, neurosensory and neuromotor disturbances, arthralgias and myalgias, heart failure, and treatment-related deaths). Despite the long dismal history of repeated failures tomeaningfully improve survival rates by aggressive combination chemotherapy with non-cross-reacting drugs, hope is nevertheless expressed that the future will be different with the new molecularly targeted agents.
However, no matter how many effective mechanistically-different anticancer agents there are, and no matter how superior their therapeutic index, cancer cell demise occurs by only two cell death pathways (necrosis or apoptosis). If the two celldeath mechanisms are attenuated by drug resistance mechanisms (e.g. p-glycoprotein and/or glutathione prevent intracellular drug levels reaching concentration levels sufficient to fully activate the necrosis pathway; caspase deletions and endogenouscaspase inhibitors prevent completion of apoptosis), these tumor cells are only sublethally injured, recover, and proliferate to kill the patient. The history of the results of these clinical trials is therapeutic equivalence between differentcombination chemotherapy "doublets" and "triplets". The repeated failures of this approach to overcoming clinical drug resistance--i.e., the lack of clinically relevant differences in overall survival-means only continuance of palliative treatment withdecisions tailored individually around such issues as differences in toxicity profiles, patients' age and performance status, and quality of life.
New agents, no matter a new molecular target or superior therapeutic index, can only kill cancer cells if there is completion of the cell death pathways through death's door. In drug-resistant cells, it is not the activation of their cell deathpathways by clinically effective anticancer agents that is at fault, but rather pathway completion to death of the cell. This reality suggests that continuance of this failed strategy utilizing only aggressive combination chemotherapy withnon-cross-reacting drug-will likely result in-to quote Yogi Berra--"deja vu all over again".
The above facts suggest the development of a treatment (co-administered with the initiation of activity in the cell death pathways by anticancer agents) that complements and augments the agent-induced initiation of activity in cell death pathwaysto bring the pathway to completion; namely, to cell death. That treatment, focused on severe ATP depletion, has been developed and proven at the preclinical level, and is about to undergo validation by clinical trial with clinical supplies of theATP-depleting regimen provided by the NCI RAID grant mechanisms.
Heterogeneous neoplastic cell populations likely contain cancer cells of variable sensitivity to the anticancer agents. Less sensitive cells would not receive enough damage to reduce ATP to low levels sufficient to cause necrotic death. Wehypothesized that biochemical modulation to further depress ATP to lower lethal-inducing levels would kill these sublethally-injured cells, augment tumor regressions, and perhaps even yield some cures.
SUMMARY OF THE INVENTION
This invention provides a composition comprising a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cells, wherein at least one of the ATP-depleting agents is a mitochondrialATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
The ATP level is depleted to at least 15% of normal in cancer cells. In some embodiments, it is substantially lower than 15%. For example, the level could be 5% of normal. In a further embodiment, the level is as low as 1%.
This invention also provides a composition comprising an effective amount of a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cells, and a pyrimidine antagonist, wherein atleast one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides a method for treating a cancer subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancercells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides a method for treating a cancer subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancercells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, whereinsaid composition produces a substantially better effect than a composition without at least one of the following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor ofDe Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides a method for induction of cancer cell death comprising contacting said cancer cell with a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cellswherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides a method for induction of cancer cell death comprising contacting said cancer cell with a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cellswherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, wherein saidcomposition produces a substantially better effect than a composition without at least one of the following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of DeNovo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention provides a method for treating a cancer subject, and for the induction of cancer cell death, comprising administering to the subject a combination of ATP-depleting agents, a pyrimidine antagonist, and anticancer agent to which thetreated cancer is sensitive, at concentrations which together collectively deplete the ATP levels to at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitors, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention provides a method for treating a cancer subject, and for the induction of cancer cell death, comprising administering to the subject a combination of ATP-depleting agents, a pyrimidine antagonist, and anticancer agent to which thetreated cancer is sensitive, at concentrations which together collectively deplete the ATP levels to at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosinephosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, wherein said composition produces an effect which is substantially better than a composition without at least one ofthe following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
DETAILED DESCRIPTION OF THE INVENTION
Patent Cooperation Treaty (PCT) Application PCT/US01/46886 (International Publication Number WO 02/4720A1) discloses treatment of cancer by reduction of intracellular energy and pyrimidine. Specifically, PCT/US01/46886 highlighted the importanceof depletion of ATP in cancer therapy. The disclosure herein provides improvements over this PCT application.
This invention provides a composition comprising a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cells, wherein at least one of the ATP-depleting agents is a mitochondrialATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
The ATP level is depleted to at least 15% of normal in cancer cells. In some embodiments, it is substantially lower than 15%. For an example, the level could be 5 to 10% of normal. In another instance, the level is 1 to 4%. In a furtherembodiment, the level is as low as 1%.
This invention also provides a composition comprising an effective amount of a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cells, wherein at least one of the ATP-depletingagents is a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor, of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides the above compositions, wherein said compositions produce a substantially better effect than a composition without at least one of the following ATP depleting agents: a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
As used herein, substantially better means that the composition could deplete the intracellular energy level at least 5% better. In another embodiment, the composition is 5% to 100% better. In a separate embodiment, it is 10% to 100% better. In a further embodiment, it is 15% to 100% better. In a still further embodiment, the composition is 20% to 100% better. In a further embodiment, it is 25% to 100% better. In another embodiment, it is 30% to 100% better. In a separate embodiment, itis 35% to 100% better. In another embodiment, it is 40% to 100% better. In a further embodiment, it is 45% to 100% better. In another embodiment, it is 50% to 100% better. In a still further embodiment, it is 55% to 100% better. In a separateembodiment, it is 60% to 100% better. In a still separate embodiment, it is 65% to 100% better. In another embodiment, it is 70% to 100% better. In a separate embodiment, it is 75% to 100% better. In another embodiment, it is 80% to 100% better. Ina separate embodiment, it is 85% to 100% better. In a further embodiment, it is 90% to 100% better. Finally, the composition may be 95% to 100% better.
To achieve "substantially better than the composition without", it is not necessary that the depleting agents used be more than one agent. In fact, a single agent may be capable of performing such better effect.
The above compositions may further comprise a pyrimidine depleting agent. Further, the above compositions may comprise an anticancer agent to which the cancer is sensitive. In a still further embodiment, the anticancer agent is at approximatelyhalf the maximum tolerated dose.
These agents stated hereinabove may be a single agent but with more than one function. For example, an ATP-depleting agent may also be an anticancer agent.
The ATP-depleting agents include but are not limited to 6-methylmercaptopurine riboside (MMPR), 6-Aminonicotinamide (6-AN) or alanosine (AL).
The above compositions may further comprise N(phosphonacetyl)-L-aspartic acid (PALA). In a separate embodiment of the compositions, it comprises 3-bromopyruvic acid.
The above composition further comprises dehydroepiandrosterone (DHEA), oxythiamine (OT) or in combination thereof.
In an embodiment, the composition further comprises 6-Aminonicotinomide (6-AN).
The above composition may further comprise a cytokine. In an embodiment, the cytokine is G-CSF.
This invention also provides a pharmaceutical composition comprising the above composition and a pharmaceutically acceptable carrier.
For the purposes of this invention, "pharmaceutically acceptable carriers" mean any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules and capsules, etc. Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.
This invention also provides the combination of ATP-depleting agents and the anticancer agent to treat drug-resistant cancer cells. In an embodiment, the dose of the anticancer agent is at approximately half of the maximum tolerated dose.
This invention also provide a method for treating a cancer in a subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancercells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside or combination thereof.
This invention also provides a method for treating a cancer subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancercells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitor, methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, and said composition produces a substantially better effect than a composition without at least one inhibitor of the following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosinephosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside or a combination thereof.
This invention also provides the above methods wherein the agents further comprise a pyrimidine-depleting agent.
The above methods may further comprise an anticancer agent. In another embodiment, the cancer is clinically sensitive to the employed anti-cancer agent.
In a separate embodiment, the anticancer agent is at approximately half of the maximum tolerated dose.
This invention also provides a method for induction of cancer cell death comprising contacting said cancer cell with a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cellswherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention also provides a method for induction of cancer cell death comprising contacting said cancer cell with a combination of ATP-depleting agents at concentrations which deplete the ATP level to at least 15% of normal in cancer cellswherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, wherein saidcomposition produces a substantially better effect than a composition without at least one of the following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of DeNovo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
The above methods may comprise a pyrimidine-depleting agent. In an embodiment, the above methods further comprise an anticancer agent.
In an embodiment, the cancer is clinically sensitive to the employed anticancer agent. In a further embodiment, the anticancer agent is at approximately half of the maximum tolerated dose.
This invention provides a method for treating a cancer subject, and for the induction of cancer cell death, comprising administering to the subject a combination of ATP-depleting agents, a pyrimidine antagonist, and anticancer agent to which thetreated cancer is sensitive, at concentrations which together collectively deplete the ATP levels to at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitors, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention provides a method for treating a cancer subject, and for the induction of cancer cell death, comprising administering to the subject a combination of ATP-depleting agents, a pyrimidine antagonist, and anticancer agent to which thetreated cancer is sensitive, at concentrations which together collectively deplete the ATP levels to at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosinephosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, wherein said composition produces an effect which is substantially better than a composition without at least one ofthe following ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
In an embodiment of the above methods, the anticancer agent is at approximately half of the maximum tolerated dose.
The ATP-depleting agents used in the above methods include but are not limited to 6-methylmercaptopurine riboside (MMPR), 6-Aminonicotinamide (6-AN) and alanosine (AL).
In an embodiment of the above methods, the methods further comprise N-(phosphonacetyl)-L-aspartic acid (PALA).
In an embodiment of the above methods, the method further comprises 3-bromopyruvic acid.
In an embodiment of the above methods, the method further comprises N-(phosphonacetyl)-L-aspartic acid (PALA).
In an embodiment of the above methods, the method further comprise dehydroepiandrosterone (DHEA), oxythiamine (OT) or in combination thereof.
In an embodiment of the above methods, the method further comprises 6-Aminonicotinamide (6-AN).
In an embodiment of the above methods, the method further comprises a cytokine. The cytokine includes but is not limited to G-CSF.
This invention also provides a method for killing drug-resistant cancer cells comprising contacting the combination of ATP-depleting agents and the anticancer agent. In an embodiment, the dose of the anticancer agent is at approximately half ofthe maximum tolerated dosage.
This invention provides a method for treating drug-resistant cancer cells comprising contact the said cancer with a combination of ATP-depleting agents and an anticancer agent.
In an embodiment the dose of said anticancer agent is at approximately half of the maximum tolerated dose.
This invention provides the above method, wherein the ATP level is depleted to at least 15% of normal in cancer cells and at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosinephosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof.
This invention further provides the above described method wherein the ATP level is depleted to at least 15% of normal in cancer cells and at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, or a combination thereof, within and said composition produces an effect which is substantially better than a compositionwithout at least one of the ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside or a combinationthereof.
Finally, this invention provides method for induction of cancer cell death comprising contacting said cancer cell with an agent capable of inducing necrosis in cancer cells. In an embodiment the agent is an ATP-depleting agent. This method mayinclude a pyrimidine-depleting regimen, or a combination thereof.
The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit theinvention as described herein, which is defined by the claims which follow thereafter.
Experimental Details
Map
An ATP-reducing regimen, MAP--an acronym for the combination of 6-methylmercaptopurine riboside, MMPR, +6-aminonicotinamide, 6-AN, +N-(phosphonoacetyl)-L-aspartic acid, PALA)--when co-administered with anticancer agents; e.g., Doxorubicin A(Adriamycin)--enhances experimental tumor regressions in vivo via the necrosis cell death pathway (1). MMPR is an inhibitor of de novo purine biosynthesis that limits adenine supplies for ATP production. (2) 6-AN is an inhibitor of the generation ofATP in the glycolysis pathway (3-6). The cell-killing ATP threshold is 15% of basal levels, and lower (7-8). Since multiple biochemical pathways contribute to the synthesis, generation and maintenance of ATP, a combination of two ATP-depleting agents,MMPR+6-AN, is co-administered with effective anticancer agents to concomitantly block several of the ATP-producing pathways to achieve the ATP-depleting objective of .ltoreq.15% of normal. PALA, a de novo pyrimidine biosynthesis inhibitor, can, at lownon-toxic dosages, selectively lower pyrimidine nucleotide levels in tumors (9). MMPR+6-AN can markedly lower ATP levels to an average of 15% of normal in murine mammary cancers (1). In the presence of such severely depleted ATP levels the salvagepathway formation of pyrimidine di-and tri-phosphates can be inhibited at the kinase step. Biochemical analysis shows that MAP-treated tumors are severely depleted of two vitally needed metabolites: the UTP pools to 14% of normal (10), and ATP to 15% ofnormal (1).
Necrosis cell death ensues because intracellular ATP levels at 15% of basal or lower can not sustain cellular homeostasis (7-8, 11-12). Indeed, intracellular ATP levels determine cell fate by apoptosis or necrosis (11) and can effect a "switch"in the cell death mode between apoptosis and necrosis (12). There are many references that anticancer agents that usually cause cell death by apoptosis, when blocked by exogenous or endogenous caspase inhibitors, or by manipulation (i.e., depletion) ofenergy metabolism, "switch" to necrosis cell death due to severe ATP depletion (references summarized in Table 1).
TABLE-US-00001 TABLE 1 EVIDENCE IN NON-DRUG RESISTANT CELLS THAT APOPTOTIC INDUCERS CAN BE MADE TO "SWITCH" TO THE NECROSIS CELL DEATH PATHWAY Apoptotic Apoptosis No Inducer Cells Blocker Result Reference 1 EGF Pancreas Caspasel- Apoptosis toFEBS inhibitor non-apoptotic Lett. `necrotic`- 491:108-108, like deaths 2001 2 Staurosporin, Jurkat Inhibitor of Apoptosis to Cell Death VP-16, Act Lymphoma caspases necrosis Differ. D T-cell 5:298-306, 1998 3 TNF L929 and Inhibitor of Enhanced J. Exp. HeLa cells caspases necrosis Med, 187:1477-1485, 1998 4 Staurosporin, Human T Pre-emptied Apoptosis to Cell Death CD95 cells cells of ATP necrosis Differ. stimulation 4:435-442, 1997 5 Fas mAb Jurkat T ATP Apoptosis to Cancer stimulation cells, HeLadepletion necrosis Res. 57:1835-1840, 1997 6 Dexamethasone B-lymphocytes Inhibitor of Apoptosis to FEBS Lett. caspases necrosis 425:266-270, 1998 7 Dexamethasone Thymocytes Inhibitor of Apoptosis to Oncogene Etoposide caspases necrosis 15:1573-1581,1997 8 Staurosporin, Human T Per-emptied Apoptosis to J. Exp. CD95 agonist cells cells of ATP necrosis Med. 185:1481-1486, 1997 9 Deoxycholic HCT 116 Overexpression Apoptosis to Cancer acid cells on of bcl-2 necrosis Lett. (activates or PKC152:107-113, Fas) 2000 10 H.sub.2O.sub.2 HN U937 Increasing Apoptosis to Exp. Cell cells H.sub.2O.sub.2 necroses Res concentration 221:462-469, 1995 11 Bax Jurkat cell Inhibitor of Apoptosis to Proc Natl caspases "non- Acad Sci. apoptotic" 93:14559,death 1996 12 Camptothecin Leukemia U- Inhibitor of Apoptosis to Cancer 937 cells caspases necrosis Res. 59:3565-3569, 1999 13 Anti-F25 L 929 cells Inhibitor of Apoptosis to J. Exp. Abs caspases necrosis Med. 188:919-930, 1998 14 Anti-F25 JurkatInhibitor of Apoptosis to J. Cell Abs cells caspases necrosis Biol. 143:1353-1360, 1998 15 TNF-a Mouse 3T3 Inhibitor of Normally J. Virol. fibroblasts caspases resistant; 74:7470-7477, switch to 2000 necrosis
The reason is that anticancer agents usually damage DNA, either directly or indirectly, and thereby activate the two principal pathways of cell death, necrosis and apoptosis, simultaneously in the same cancer cell. Only one death pathwayprevails in the cells, as determined by intracellular conditions. However, the two modes of cell death can occur in different cells simultaneously in the same tumor exposed to the same anticancer agent. One reason is that different drug concentrationsreach different cancer cells; low concentrations induce apoptosis and high concentrations cause necrotic cell death (13). If the anticancer drug's intracellular concentration is high, the drug-DNA "hit" is then of sufficient magnitude to stronglyactivate poly (ADP-ribose) polymerase (PARP), and PARP rapidly destroys and depletes glycolytic NAD severely inhibiting the glycolytic production of ATP (1). The resulting severe depletion of ATP causes cell death by necrosis (1). And, although lowconcentrations of anticancer agents do not damage DNA sufficiently to cause the marked lowering of ATP that results in necrotic cell death, there is still a reduction of ATP. Hence, all effective anticancer agents always cause ATP reduction, and areconsidered part of our ATP-depleting regimen. An important part, because as "selective" agents for cancer cells, effective anticancer agents preferentially reduce more ATP in cancer cells than normal cells. The agents thereby create a therapeuticopportunity for co-administration of biochemical modulators (i.e., the concomitant addition of an ATP-depleting regimen-e.g., MAP) to further reduce cancer cell ATP to the severely low levels (i.e., .ltoreq.15% of normal) that kill, and the cells die anecrotic death.
MAP plus each of ten mechanistically different anticancer drugs at half-MTD (doxorubicin, paclitaxel, docetaxel, cisplatin, phenylalanine mustard, mitomycin C, cyclophosphamide, etoposide, 5-fluorouracil, and radiotherapy) has been demonstratedin vivo to be safe and to cause significantly greater tumor-regressions than the MTD of each anticancer agent alone in hundreds of animals bearing advanced spontaneous and first generation murine breast cancer (references summarized in 1), as well as inmurine leukemia and colon tumors. "Cures" (25%) were produced with MAP+radiotherapy in tumor-bearing mice followed for over 380 days (14). The improved therapeutic results were all produced by MAP+each anticancer agent at half-MTD, strongly suggestingthat a marked reduction of the toxic side effects of usual MTD clinical cancer chemotherapy will be obtained in future clinical trials.
Spontaneous murine breast cancers have drug-resistant cancer cells as part of their heterogeneous neoplastic cell population just as spontaneously-arising human cancers do. The fact that the number of tumor regressions induced by the combinationof MAP+ anticancer agents were always significantly greater than the regressions produced by each anticancer agents alone at MTD suggested that the additional cell kills occurred in drug-resistant cancer cells. Drug-resistant cancer cells are known tobe frequently blocked in apoptosis, but not known to have blocks per se in the necrosis cell death pathway. It is likely that drug-resistant cells were killed in the spontaneous tumor because the ATP-depleting strategy is specifically targeted at thelatter pathway. However, to obtain a clearer focus of the effect of the ATP-depleting strategy plus an anticancer agent on drug-resistant cancer cells, treatment was done on multi-drug-resistant, overexpressed p-glycoprotein NCI-AR-Res(Adriamycin-resistant) human mammary carcinoma xenografts (15). (See Table 2, below)
MAPAL
As previously noted, the ATP depletion induced in murine mammary tumors by (MMPR+6-AN) of MAP averaged 15% of normal, the cytocidal threshold level of ATP shown to be insufficient to sustain cell viability (7-8). Hence, some individual tumorcells have ATP levels <15% of normal, are killed, and the tumors regress. Other MAP-treated tumors have reduced ATP levels >15% of normal and these tumors are only inhibited in their growth (7). Obviously if the (MMPR+6-AN) induced ATP tumorlevel had averaged lower than 15% of normal, there would be more tumor cells killed, and more tumor regressions. A stronger ATP-depleting regimen was therefore sought. Alanosine (AL), an inhibitor of de novo adenosine monophosphate synthesis (16), hadbeen previously shown to add to MMPR's reduction of ATP (17). In the latter studies, MMPR alone lowered ATP levels to 49% of normal, and alanosine (AL) alone to 63% of normal, but in combination they decreased ATP to 34% of normal. This strikingsynergistic reduction of ATP when combined suggested that the addition of AL to MAP(acronym: MAPAL) might safely reduce the average tumor ATP levels still lower than the average 15% of normal induced by MAP. We have not the funding opportunity tocompare in the same tumors the average % of induced ATP depletion by MAPAL as compared to MAP. However, the evaluation of MAPAL+an antitumor agent to MAP+the same anticancer agent against advanced mammary tumors in mice showed MAPAL to betherapeutically more effective than MAP, and with no mortality and only an 8% weight loss (unpublished studies).
MAPAL, therefore, was the ATP-depleting regimen chosen to be evaluated against NCI-AR-RES (Adriamycin-resistant) xenografts, and the therapeutic results are summarized in Table 2.
TABLE-US-00002 TABLE 2 THE ADDITION OF AN ATP-DEPLETING REGIMAN, MAPAL, ENABLES LO-DOSE ADRIAMYCIN TO KILL DRUG-RESISTANT CANCER CELLS IN ADRIAMYCIN-RESISTANT HUMAN MAMMARY CARCINOMA XENOGRAFTS BY THE NECROSIS CELL DEATH PATHWAY Treatment BodyWt p- Group.sup.a n.sup.c change.sup.c % PR.sup.c % Value.sup.d 1. Control 20 +6.6 0 2. Adria.sub.10 57 -7.8 0 3. MAPAL.sup.b 76 -3.8 18 (14/76) 0.0003 4. MAPAL .fwdarw. 77 -7.9 48 (37/77) <.0001 21/2 hr.fwdarw. Adria.sub.5 5. MAPAL +Adria.sub.5 52 -9 46 (24/52) <.0001 (SIMULTANEOUSLY) .sup.aPooled experiments (99, 103, 108, 112, 114, 115, 116, 117): Nude mice (nu/nu) with NCI-AR-RES mmamry carcinoma xenografts averaged 100 mg when treatment initiated. Indicated treatmentadministered in three cycles with a 14 day interval between cycles. .sup.bMAPAL = 6-methylmercaptopurine riboside (MMPR.sub.150) + 6-aminocotinamide (6-AN.sub.6) + PALA.sub.100 + alanosine (AL.sub.250); subscript = mg/kg. PALA 17 hrs prior to (MMPR + 6- AN + AL) 2.5 hrs prior to Adria. All agents i.p., except Adria i.v. .sup.cTherapeutic observations recorded one week after the third cource (35 days after initiating therapy.) . PR = .gtoreq.50% decrease in tumor volume .sup.dCompared toAdria.sub.10Group 2. Comparing Group 3 VS Group 4 =<.0001; VS Group 5, 0.0011
Comment--The combination of MAPAL and Adria (48% PR or 46% PR) is more effective in killing cells than just MAPAL (18% PR), and the use of only half-MTD of Adria in the presence of MAPAL (48% PR or 46% PR) is remarkably more effective in killingdrug-resistant cancer than the full dose(MTD) of Adria (0% PR). The striking positive therapeutic results--almost a 50% P.R. rate using a 50% lower dose of Adria (with MAPAL) compared to a 0% P.R. rate with high dose Adria(alone)--auger well for anamelioration of the toxic side effects of high dose Adria in clinical trial along with greater therapeutic results.
It is stressed that there is no difference between the PR rates of Group 4, MAPAL 21/2 hours prior to Adria, vs. Group 5, MAPAL simultaneously administered with Adria; namely, 48% P.R. vs. 46% P.R., respectively. It is therefore clear thatthe time differential (21/2 hrs.) did not affect the results; suspicion had been raised that the ATP-dependent p-glycoprotein drug efflux pump might be adversely affected by prior(21/2 hr) ATP reduction in Group 4 as the reason for previously publishedpositive therapeutic results with that time interval.
Molecular biology studies (Table 1) demonstrate that the necrosis cell death pathway can fully function (i.e., kill) to bypass a blocked apoptosis pathway, or can be fully evoked following manipulation of energy metabolism to kill cells. Thefindings in these studies in non-drug resistant cells appear relevant to drug-resistant cells, which are blocked in apoptosis but not known to have blocks per se in the necrosis cell death pathway. Thus, if their apoptosis pathway is blocked but theirnecrosis pathway is "open", a combination of anticancer agents and ATP-depletion agents should be able to lower ATP to cytocidal levels and selectively kill drug-resistant cells. The findings in these fifteen referenced studies imply that the necrosiscell death pathway could be the therapeutic opportunity to overcome drug-resistant cancer cells.
The primary similarity in the treated cancer cells of these molecular biology studies to drug-resistant cells is that apoptosis activity is prevented in both. In the drug-resistant cells triggered apoptosis cannot proceed to completion becauseof inherent multiple drug resistance factors; e.g., endogenous caspase inhibitors (so-called IAPs), genetic deletions of caspases, over-expressed anti-apoptotic bcl-2, mutations in p53 and other processing units in the apoptotic program, or in someindividual cancer cells the presence of all of these genetically-induced resistance factors. Thus, apoptosis may be triggered (initiated), but not completed, by drug-treatment in the many above-noted molecular biology studies as well as in thedrug-resistant cancer cells.
However, there is a significant difference in comparing necrosis pathways. In the molecular biology studies with non-drug resistant cells there are no resistance factors (i.e., obstacles) leading to the necrosis pathway, and the usual apoptosiscell death mode is readily "switched" to necrosis. In contrast, drug-resistant cells can have resistance factors (e.g., drug efflux enzymes) that only allow for low drug concentrations that effect only partial completion of the necrosis pathway). Highintracellular drug concentrations are required for cell death by necrosis. Necrosis will not occur unless the drug-target "hit" (usually to DNA) is of sufficient intensity to strongly activate PARP so as to result in a rapid and severe depletion of ATPto cell-killing levels (.ltoreq.15% of normal). In drug-resistant cancer cells, the drug-induced necrosis pathway is initiated but does not proceed to completion (i.e., to death) because the drug-resistance factors of over-expressed drug efflux pumps(e.g., p-glycoproteins), and/or intracellular drug detoxifiers (e.g., glutathione), lower the intracellular drug level and diminish the drug-target interaction. Hence, although the result is still reduction of ATP, it is not low enough to belethal-inducing (i.e., .ltoreq.15% normal ATP). The necrosis pathway is only partially activated, and the ATP depletion does not fall to levels sufficient for cell-killing. Reductions of ATP to above the cell-killing threshold will only cause transientinhibition of cancer cell proliferation followed by complete recovery (7). Thus, following present chemotherapy, drug-resistant cancer cells are only sub-lethally-injured, recover, and grow back to kill the patient.
The molecular biology references (Table 1) suggest that a substantial but non-lethal degree of ATP depletion always occurs in anticancer agent-induced sub-lethally-injured drug-resistant cancer cells because there is only initiation, but notcompletion (i.e., to death) of the necrosis cell death pathway. Thus, since most effective DNA-damaging anticancer agents are themselves ATP-reducing agents, the further reduction of ATP to the ATP lethality threshold by an ATP-depleting regimen shouldkill these drug-resistant cells--and does (Table 2). Previous preclinical studies successfully overcoming one or another of the many drug-resistant factors have foundered in the clinical arena because drug-resistant factors are multifactorial. However,the novel ATP-depleting strategy is not aimed at any of the drug-resistant factors, but bypasses them all by directly killing drug-resistant cancer cells through their vulnerable necrosis cell death pathway.
PALM-Bromopyruvate(BrPA)
In non-drug-resistant tumors, Geschwind et al (18) have reported that a potent ATP-depleting agent (3-bromopyruvic acid, or BrPA), when delivered by "direct" intraarterial administration to liver-implanted rabbit tumors, eradicates nearly allviable cancer cells by necrosis, and also without harm to normal cells or to the animals. The absence of mortality and serious toxicity further obviates the notion that severe intra-tumor ATP depletion must be toxic to both normal and cancer cells.
No ATP measurements were reported in the successful cancer cell-killing therapy with BrPA as a single agent. The very high concentration of BrPA achieved by the "direct" arterial delivery apparently depleted ATP in the "directly"-treated cancercells to cell-killing ATP levels because the same dose of BrPA by systemic i.v. administration only inhibited tumor growth, apparently due to a diluted concentration by the whole blood volume only lowering ATP to levels above the cell-killing ATPthreshold, levels shown to effect only inhibition of tumor growth. The level of ATP depletion is all important as regards the therapeutic result (ie, death of cells vs. their transient inhibition). This understanding provides insight for the pastfailures and present success of ATP-depletion.
Previous attempts at ATP-depletion as therapy have failed due to the lack of understanding of how it would be effective in killing cancer cells. Only recently has there been data establishing that there is a cell-killing ATP threshold of 15% ofbasal levels and below(7-8), and that attaining this severe lowering of intracellular ATP levels in cancer cells normally requires the concomitant administration of multiple ATP-depleting agents(1).
In line with our planned step by step development of a "best" multiple-ATP-depleting regimen, the incorporation of "selective" antimitochondrial agents (e.g., F16; 19) had been programmed for eventual inclusion in the ongoing development of thenew and promising ATP-depleting therapeutic strategy. Although BrPA has not been reported as "selective", it was noteworthy in reducing cellular ATP production via the inhibition of both glycolysis and oxidative phosphorylation (18). It was thereforesubstituted for the glycolysis inhibitor, 6-AN, in MAPAL. Without 6-AN, MAPAL includes only PALA+Alanosine (AL)+MMPR (acronym: PALM). PALM+BrPA, or PALM-BrPA, inhibits adenine biosynthesis, glycolytic and mitochondrial ATP production. PALM-BrPA+Adria.sub.5 vs MAPAL+Adria.sub.5 were compared in therapeutic studies in multi-drug-resistant NCI-AR-RES (Adriamycin-resistant) human mammary tumor xenografts. Table 3 pools the data of four experiments.
TABLE-US-00003 TABLE 3 PALM-BRPA IS SIGNIFICANTLY SUPERIOR TO MAPAL IN ENHANCING ADRIAMYCIN-DRUG-RESISTANT CANCER CELL KILL. PALM-BRPA INDUCES SEVERE ATP REDUCTION (BY INHIBITING ADENINE BIOSYNTHESIS, GLYCOLYTIC AND MITOCHONDRIAL ATPPRODUCTION) THAT ENABLES LO-DOSE ADRIAMYCIN TO KILL ADRIAMYCIN-RESISTANT CELLS IN HUMAN MAMMARY XENOGRAFTS BY THE NECROSIS CELL DEATH PATHWAY. Treatment Body Wt Deaths.sup.c PR.sup.c Group.sup.a n.sup.c change.sup.c % % % p-Value 1. Control 8 +5 0 2. Adria.sub.10 15 -6 0 3. MAPAL + Adria.sub.5 41 -8 4 34 4. PALM + BrPA.sub.10 + 42 -13 0 62 .sup.d 0.01% Adria.sub.5 .sup.aFour pooled experiments (116, 117, 121, 122): Nude female mice with NCI-AR-Res human mammary carcinoma xenografts averaging 100 mgwhen treatment initiated. Indicated treatments were administered in 3 cycles with a 14 day interval between cycles. .sup.bMAPAL = MMPR.sub.150 + 6-AN.sub.6 + PALA.sub.100 + AL.sub.250. PALM = PALA.sub.100 + AL.sub.250 + MMPR.sub.150. BrPA =3-Bromopyruvate.sub.10. All agents i.p., except BrPA and Adria. i.v. Subscripts = mg/kg. .sup.cTherapeutic observation one week after the third course (35 days after initiating therapy). PR = .gtoreq.50% reduction in tumor size compared to tumorsize when treatment initiated. PR % = number PR per group / surviving animals per group .times. 100. .sup.dCompared to Group 3, MAPAL + Adria.sub.5.
Comment--As previously (Table 2), the MTD of Adria, 10 mg/kg, on this Adriamycin-resistant tumor is without tumor-regressing effect (0% PR), whereas Adria. at 1/2 MTD in the presence of MAPAL causes an impressive number of partial tumorregressions in drug-resistant cells (34% PR), and PALM-BrPA+Adria(1/2 MTD) is remarkably much more effective (62% PR). With only these four experiments completed, the PALM-BrPA data is considered preliminary. Nevertheless, PALM-BrPA+Adria.sub.5 appearsto have doubled (62%) the PR rate over MAPAL+Adria (34%). We have not had the funding opportunity to compare the degree of ATP depletion. Both combinations inhibit adenine supplies (i.e., by MMPR), both inhibit adenosine monophosphate biosynthesis(i.e., by AL), and both are glycolytic inhibitors, although by different mechanisms--PALM-BrPA by BrPA, and MAPAL by 6-AN. Although one may be a slightly stronger glycolytic inhibitor, the major difference clearly is that only PALM-BrPA is also amitochondrial ATP inhibitor. Therefore, it could be anticipated that PALM-BrPA would be the stronger ATP depleter, and the therapeutic results confirm this expectation.
MAP with two ATP depleters effected a tumor ATP average to the cytocidal ATP threshold of 15% of normal (1). MAPAL with three ATP depleters apparently further reduced the average ATP level below this critical cytocidal ATP threshold level. PALM-BrPA with four ATP depleters apparently reduced this critical ATP average still more. Thus, with each incremental decrease in ATP depletion below the cytocidal threshold of 15% of normal, more drug-resistant cancer cells are killed.
Combining a number of ATP-depleting agents sometimes raises concern for anticipated toxic side-effects in normal tissues. However, cancer cells are apparently preferentially vulnerable to ATP depletion, and note that the markedly increasedtumor-regressing results of PALM-BrPA, which combines four ATP-depleting components, is not accompanied by gross damage to normal tissues, or mortality.
Other ATP Depleters That Could Contribute to an ATP-Depleting Regimen
A. Inhibitors of Mitochondrial ATP--There are a number of mitochondriotoxic small molecules that selectively accumulate in the mitochondria of tumor cells, compromise their functional integrity, have antiproliferative activity, and can cause asignificant depletion of ATP pools. F16 is a small molecule that belongs to this group of compounds that have cancer cell mitochondria as a selective target (19). Although only tumor growth inhibitory as a single agent in vivo, its selectivity and lowtoxicity suggests that it could make a substantial contribution to ATP depletion as a component of a necrosis-inducing ATP-depleting combination (e.g., MAPAL-F16). F16's selectivity appears restricted to breast cancers. There are other such "selective"mitochondriotoxic molecules that display greater diversity in accumulating in the mitochondria of other types of cancer (20-21), and these should be evaluated in a multi ATP-depleting combination. B. Inhibitors of Methylthioadenosine Phosphorylase(MTAP)--MTAP is an important intracellular salvage enzyme for adenine (and methionine). Cancer cells that are deficient in MTAP are not able to salvage adenine (and methionine), and are markedly sensitive to inhibitors of the de novo synthesis ofadenine nucleotides, especially alanosine which blocks de novo AMP synthesis (22). An inhibitor of MTAP would likely be a useful component of a multi-ATP-depleting combination, because MTAP plays a critical role in recycling adenine moieties fromS-adenosylmethionine, derived originally from ATP, back to adenine nucleotide pools (23). C. Inhibitors of De Novo Purine Synthesis other than 6-Methylmercaptopurine Riboside (MMPR)--all tumors contain genetic instability and alterations, includingchromosome losses (i.e., gene deletions) and gains (i.e., overexpressed genes). (24) This genetic instability is reflected in the heterogeneity seen within individual tumors and among tumor of the same type.
Against this background, it is not surprising that MMPR, which requires metabiotic activation by phosphorylation via adenosine kinase (25), meets with resistance in cancers devoid of adenosine kinase (26-27). Since MMPR is an important componentof MAPAL and PALM, the ATP-depleting strategy would not be effective in adenosine kinase-deficient cancer cells. Also, cancer cells with overexpressed multidrug resistance protein 4, a drug-efflux pump that transports MMPR out of the cell, would reducethe MMPR intracellular concentration, and be a resistance mechanism (28). Substitution of MMPR in the multi-ATP-depleting regimen by antifolates that target purine synthesis should be as effective as MMPR, might even be better, and should be evaluated. These novel antifolates include DDATHF (29), Agouron compound AG 2034 (30), and PDX (31-32).
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S., Purine and Pyrimidine Biochemistry, and some relevant clinical and preclinical cancer chemotherapy research. In: G. Powis and R. A. Prough (eds.), Metabolism and Action of Anti-Cancer Drugs, pp. 91-140. London: Taylor and Francis, 1987. 11. Eguchi, Y., Shimizu, S., and Tsujimoto, Y. Intracellular ATP levels determine cell fate by apoptosis or necrosis. Cancer Res., 57: 1835-1840, 1997. 12. Leist, M., Single, B., Castoldi, A. F., Kuknle, S., and Nicotera, P. Intracellular triphosphate(ATP) concentration: A switch in the decision between apoptosis and necrosis. J. Exp. Med., 185: 1835-1840, 1997. 13. Huschtscha, L. I., Anderson, C. E., Bartier, W. A., and Tattersall, M. H. N. Anti-cancer drugs and apoptosis. In: M. Lavin and D.Walters (eds.), Programmed Cell Death, the Cellular and Molecular Biology of Apoptosis, pp. 269-278. New York: Harwood Academic, 1993. 14. Koutcher, J. A., Alfieri, A., Stolfi, R. L., Devitt, M. L., Colofiore, J. R., Nord, L. D., and Martin, D. S.Potentiation of a three drug chemotherapy regimen by radiation. Cancer Res. 53: 3518-3823, 1993. 15. Scudiero, D. A., Monks, A., and Sausville, E. A. Cell line designation change: Multidrug-resistant cell line in the NCI anticancer screen. J. Natl. Cancer Inst. 90: 862, 1998. 16. Tyagic, A. K. and Cooney, D. A. Biomedical pharmacology, metabolism and mechanism of action of L-alanosine, a novel, natural antitumor agent. Adv. Pharmacal. Chemother. 20: 69-121, 1984. 17. Nguygen, B. T., ElSayed, Y. M., and Sadee, W. Interaction among the distinct effects of adenine and guanine depletion in mouse lymphoma cells. Cancer Res. 44: 2272-2277, 1984. 18. Geschwind, J-F. H., Ko, Y. H., Torbenson, M. S., Magee, C., and Pedersen, P. L. Noveltherapy for liver cancer: Direct inhibitor of ATP production. Cancer Res, 62: 3909-3913, 2002 19. Fantin, V. R., Berardi, M. J., Scorrano, L., Koromeyer, S. J., and Leder, P. A novel mitochondreotoxic small molecule that selectively inhibit tumor cellgrowth. Cancer Cell 2: 29-42, 2002 20. Modica Napolitano, J. S. and Aprille, J. R. Delocalizld lipophilic cations selectively target the mitochondria of carcinoma cells. Adv. Drug Deliv. Rev. 49: 63-70, 2001. 21. Britten, C. D., Rowinsky, E. K.,Baker, S. D., Weiss, G. R., Smith, L., staphenson, J., Rothenberg, M., Smetzer, L., Cramer, J., Collins, W., Von Hoff, D. D., and Eckhardt, S. G. A Phase 1 and pharmacokinetic study of the mitochondrial-specific chodacyanine dye analog MKT 011. Clin.Cancer Res. 6: 42-49, 2000. 22. Yu, J. Alanosine (UCSD). Curr Opin Investig Drugs 2 (11): 1623-30, 2001. 23. Williams-Ashman, H. G., Seidenfeld, J. and Galletti, P. Trends in the biochemical pharmacology of 5'-deoxy-5'-methylthioadenosine. Biochempharmacal. 31: 277-288, 1982. 24. Cahill, D. P., Kinzler, K. W., Vogelstein, B., and Lengauer, C. Genetic instability and Darwinian selection in tumors. Trends Biachem. Sci. 59-60, 1999. 25. Martin, D. S. Purine and pyrimidine biochemistry andsome relevant clinical and preclinical cancer chemotherapy research, in Metabolism and action of Anticancer Drugs (Powis, G. and Prough, A. R. ds), pp. 91-139, Taylor and Francis, London. 26. Cory, A. H., and Cory, J. G. Use of nucleoside Kinasedeficient mouse leukemia L1210 cell lines to determine metabolic routes of activation of antitumor nucleoside analogs. Adv Enzyme Regul 34: 1-12, 1994. 27. Young, I., Young, G. L., Wiley, J. S., and van der Weyden, M. B. Nucleoside transport andcytosine arabinoside (ara C) metabolism in human T lymphoblasts resistant to ara C, thymidine and 6-methyemercap to purine riboside. Eur J Cancer Clin Oncol 21(9): 1077-82, 1985. 28. Wielinga, P. R., Reid, G., Challa, E. E., van der Heijden, I., vanDeemter, L., De Haas M., Mol, C., Kuil, A. J., Groeneveld, E., Schuetz, J. D., Brouwer, C., De Abreu, R, A., Wijnholds, J., Bejnen, J. H., and Borst, P. Thiopurine metabolism and identification of the Thiopurine metabolites transported by MRP4 and MRP5overexpressed in human embryonic kidney cells. Mol. Pharm, 62: 1321-1331, 2002. 29. Bronder, J. L. and Moran, R. G. antifolates targeting purine synthesis allow entry of tumor cells into S phase regardless of p53 function. Cancer Res. 62: 5236-5241,2002. 30. Bissett, D., Mcleod, H. L., Sheedy, B., Collier, M., Pithavala, Y., Paradiso, L., Pitsiladas, M. and Cassidy, J. Phase 1 dose-escalation and pharmacokinetic study of a novel folate analogue A G 2034. Br. J. Cancer 84: 308-312, 2001. 31. Sirotnak, F. M., De Graw, J. I., Colwell, W. T., and Piper, J. R. A new analogue of 10-deazaminopterin with markedly enhanced curative effects against human tumor xenografts in mice. Cancer Chemother Pharmacal 42: 313-318, 1998. 32. Krug, L. M., Ng,K. K., Kris, M. G., Miller, V. A., Tong, W., Heelan, R. J., Leon, L., Leung, D., Kelly, J., Grant, S. C., and Sirotnak, F. M. Phase I and pharmacokinetic study of 10-propargyt-10-deazaminopterin a new antifolate. Clin Cancer Res: 3493-3498, 2000. ATP-Depleting Combination
(i.e., the ATP-depleting combination that, when co-administered with an anticancer agent effective against the target cancer, achieves cell-killing levels of ATP in drug-resistant cancer cells with the least toxicity to white blood cells andnerves.) 1. The initially employed ATP-depleting agents--6methyl-mercaptopurine riboside (MMPR)+6-aminonicotinamide (6-AN)--were administered in combination with N-(phosphonacetyl)-L-aspartic acid (PALA), and received the acronym: MAP. MAP enhancedanticancer agent activity against drug-resistant cancer cells. 2. Subsequently, MMPR and 6-AN were combined with alanosine (AL), and received the acronym:MAPAL (i.e., MMPR+6-AN+PALA+AL). MAPAL proved a more effective ATP-depleting agent than MAP. Inin vivo studies in tumor-bearing animals, MAPAL, when co-administered with anticancer agents, evidenced greater anticancer activity than MAP, but with occasional evidence of greater myelotoxicity. MAPAL, nevertheless, is better than MAP. 3. Thecombination of PALA+AL+MMPR without 6-AN (acronym: PALM), when co-administered with anticancer agents, appears to produce the same or better antitumor activity than MAPAL without this occasional toxicity. MAPAL needs to be compared with PALM under thesame conditions. 4. 6-AN inhibits glycolysis (1), a desirable ATP-depleting effect since the vast majority of solid cancers depend on glycolysis as a source of ATP (2). 6-AN acts by inhibiting the oxidative portion of the Pentose Phosphate Pathway(PPP) via inhibition of the second enzyme of the PPP. DHEA (dehydroepiandrosterone) inhibits the first enzymes of the oxidated version of the PPP, and, although inferior as an inhibitor of glycolosis to 6-AN is less toxic than 6-AN. But since theglycolytic production of ATP receives substrate contributions (fructose-6-phosphate and glyceraldehyde-3-phosphate) from the PPP, the oxidative portion of the PPP, DHEA will likely produce the same inhibition of glycolysis when administered incombination with an inhibitor of the non-oxidative portion of the PPP, oxythiamine (OT). Thus, PALM+DHEA+OT; acronym: PALMDOT.
Nevertheless, a greater blockade of the PPP might be accomplished by inhibiting both the first and the second enzymes of the oxidative portion of PPP with DHEA+6-AN administration together with simultaneous inhibition of the non-oxidative portionof the PPP by OT; acronym: MAPALDOT. (If MAPALDOT is myelosuppressive, it may be made safe by a co-administration of G-CSF while affecting greater ATP depletion than PALMDOT.) PALMDOT and MAPALDOT need comparison.
Evaluation of the Differences Between ATP-Depleting Regimens of MAPAL, PALM, PALMDOT and MAPALDOT
Tumor Differences--Different tumor types (e.g., breast cancer vs. ovarian cancer vs. pancreatic cancer) may differ materially in the quantitative contribution of ATP they receive from different ATP-producing metabolic pathways (e.g., fromglycolysis as compared to de novo purine metabolism). If the major contribution is, for example, from de novo purine synthesis, then it may be best to employ PALM as the ATP-depleting regimen, for PALM has two inhibitors of de novo purine synthesis, AL(alanosine) and MMPR (6-methyl-mercaptopurine riboside), and no antiglycolysis component. However, while there are exceptions, the vast majority of solid cancers depend on glucose through glycolysis as an energy source, and it would be imprudent toneglect this information. Hence, there should be evaluation of MAPAL, which contains not only MMPR and AL, but also has 6-AN, a proven inhibitor of glycolysis via the oxidative portion of the Pentose Phosphate pathway (PPP). But 6-AN may havetoxicities that are non-pertinent to the goal of ATP depletion (such as rare neuroparalyis) and, therefore, it may be best to use DHEA, a less toxic inhibitor of the oxidative PPP. DHEA may be inferior to 6-AN as an inhibitor of glycolytic ATPproduction, but DHEA and 6-AN have not been previously compared in regard to reduction of glycolytic ATP, and the combination of DHEA+an inhibitor for the non-oxidative portion of the PPP, OT (oxythiamine), has not been evaluated (in combination) asregards inhibition of glycolytic ATP production.
Greater understanding of the above potential interrelationships may be obtained by comparative studies on three different cancers (the human breast cancer xenograft resistant to Adriamycin, the NCI/Adr-Res; the cisplatin (DDP)-doxorubicin-resistant human ovarian cancer xenograft, the SKOV-3; and the S2 (TXT), a taxotere (TXT)-resistant human pancreatic cancer xenograft) as follows: 1. Controls 2. Cisplatin (DDP) MTD 3. Doxorubicin (DOX) MTD 4. Taxotere (TXT) MTD 5. MAP6. MAPAL 7. PALM 8. PALMDOT 9. MAPALDOT 10. DDP half-MTD 11. DOX half-MTD 12. TXT half-MTD 13. MAP+DDP half-MTD; +DOX half-MTD; TXT half-MTD 14. MAPAL+DDP half-MTD; +DOX half-MTD; +TXT half-MTD 15. PALM+DDP half-MTD; DOX half-MTD; +TXT half-MTD16. PALMDOT+DDP half-MTD; +DOX half-MTD; +TXT half-MTD 17. MAPALDOT+DDP half-MTD; +DOX half-MTD; +TXT half-MTD (The above 27 groups (10 tumor-bearing animals per group) cannot, practically-speaking, be included in a single transplant experiment, butcan be judiciously covered in a number of smaller experiments.)
Myelotoxicity Differences--None of the above three anticancer agents alone at half-MTD, nor the various ATP-depleting regimens alone, cause myelosuppression in their tumor-bearing mice, but in combination (as in groups 13-17) occasionally do,including a rare fatality which can be prevented by either pyruvate administration, or omission of 6-AN, as in PALM. The indirect evidence, (myelosuppression only in combination, prevention by omission of 6-AN, or administration of pyruvate) suggests anadverse effect to a metabolic step of the glycolysis pathway in either the progenitor bone marrow cells or the peripheral blood leucocytes (PBL). Therefore, before treatment, and at intervals (24, 48, 72, and 96 hours) after treatment, bone marrow andPBL will be analyzed for changes in various steps of glycolysis and the PPP. An understanding of the changes may make it possible to identify a biomarker in glycolysis that will predict which tumor-bearing animal (i.e., patient) will undergo severeneutropenia and make possible early G-CSF administration for prevention as opposed to treatment by G-CSF. Such a biomarker in PBL might be glucose-6-phosphate dehydrogenase, known to be important for cell growth and in cell death(3-4).
The elaborate analysis of "best" ATP-depleting regimens, and control of PBL toxicity, detailed above is warranted by the importance of the drug resistance problem. Combination chemotherapy has been proven to cure the heterogeneity of a few typesof cancer (e.g., testicular cancer), so there is clear evidence that it is possible to cure cancer with chemotherapy. But chemotherapy fails to cure most solid cancers because of drug resistance. Since there is strong scientific evidence in support ofthe ATP-depleting therapeutic strategy to circumvent drug resistance, the elaborate evaluation as planned above is because a careful application in the clinic of preclinical guidelines should enable clinical validation of the preclinically-provenstrategy. Success is important, for the circumvention of drug resistance factors will clear the way for chemotherapeutic cure of many cancers.
REFERENCES
1. Street, J. C., Mahmoud, U., Ballon, D., Alfieri, A. A., and Koutcher, J. A. 13C and 31P NMR investigation of effect of 6-aminnicotinamide on metabolism of RIF-1 tumor cells in vivo. J. Biol. Chem. 271: 4113-9, 1996. 2. Dang, C. V. andSemenza, G. L. Oncogenic alterations of metabolism. Trends Biochem. Sci. 24: 68-92, 1999. 3. Tian, W-N., Braunstein, L. D., Pang, J., Stuhlmeier, K. M., Xi, Q-C., Tian, X., and Stanton, R. C. Importance of Glucose-6-phosphate dehydrogenase activityfor cell growth. J. Biol. Chem. 273: 10609-10617, 1998. 4. Tiam, W-N., Braunstein, L. D., Apse, K., Pang, J., Rose, M., Tian, X., and Stanton, R. C. Importance of glucose-6-phosphate dehydrogenase activity in cell death. Am. J. Physiol. 276 (CellPhysiol. 45): C1121-C1131, 1999. An ATP-Depleting Regimen (e.g., MAPAL)+an Anticancer Agent (e.g., Taxotere) Induces Permanent Growth Arrest and a Senescence-Like Phenotype in Advanced Human Breast Cancer Xenografts (MDA-MB-468) that Results in Cure.
The induction of cell senescence, along with apoptosis, and other types of cell death (e.g., necrosis, mitotic catastrophe), can be a major response of cancer cells to cytotoxic and cytostatic agents. Thus, treatment conversion of cancer cellsto senescent permanent proliferation arrest and subsequent cell death can add an important determinant of effective treatment outcome.
Neither the anticancer agent at MTD, nor the anticancer agent at half-MTD alone, nor the ATP-depleting regimen alone, effected senescence in the breast tumor xenografts. Therefore, this identification that only the combination of theATP-depleting regimen with a moderate dose of anticancer agent can induce or facilitate senescence in tumor cells is an important finding for the improvement of cancer therapy.
Present Status of Cancer Chemotherapy and Drug Resistance
Chemotherapy is the primary treatment once cancer becomes systemic (i.e., metastasizes). Most anticancer agents damage DNA in their target cancer cells, but the post-damage responses of apoptosis, necrosis, mitotic catastrophe and senescence arethwarted in drug-resistant cancer cells. The drug resistance factors are multiple, and range from mechanisms that limit the drug-target interaction (e.g., overexpression of drug efflux pumps, as p-glycoprotein, and intracellular detoxifiers, asglutathione) to genetic disruption of the apoptotic and senescence pathways. Drug-induced senescence, although difficult to induce, is increasingly being considered an important consequence of effective treatment(2,3). Mitotic catastrophe is a form ofprogrammed cell death when the DNA-damaged cells exit a cell cycle arrest and undergo fatal endomitosis(1). It is the contribution of apoptosis to therapy-induced cell death (the "apoptosis concept" that is considered by most as the pivotal responseprogram in drug-treated tumor cells (4). And necrosis, although occasionally listed as a drug-induced response, is usually not considered.
Yet, necrosis is the cell death pathway simultaneously initiated with apoptosis by drug-induced DNA damage that, unlike apoptosis, can be restored to the completion of cell death (5). Necrosis is due to severe ATP depletion (15% of normal andbelow), a cell-killing level prevented from attainment by drug-resistant factors (5). However, ATP is nevertheless reduced due to the activation of PARP by anticancer agent-induced DNA damage (5). Thus, unlike "mitotic catastrophe" and the cell deathmode of apoptosis, which are initiated but not completed in drug-resistant cancer cells, the necrosis pathway in drug-resistant cells is partially completed (5). The anticancer agent-induced DNA damage, even though lessened by drug-resistant factors,reduces ATP and thereby "chemosensitizes" the drug-resistant cancer cell for further reduction to cell-killing ATP levels by the co-administration of an appropriate ATP-depleting regimen (5). It does not matter whether one or multiple drug-resistantfactors are involved in the limitation of the necrosis pathway because the treatment to circumvent the drug-resistant factors is aimed at bringing the product of their collective blocking action--i.e., the reduced ATP level--to cell-killing levels. Theability to kill drug-resistant cancer cells by an apoptotic independent mechanism--i.e., necrosis--has been preclinically demonstrated, and is receiving NCI support for validation of the ATP-depleting strategy by clinical trial.
As noted above, there is growing evidence that senescence of cancer cells can be induced following chemotherapy and can contribute to the success of chemotherapy (2,3). It is of great additional interest that the same ATP-depleting strategy thatenhances cancer cell death by inducing necrosis, also can induce senescence in cancer cells in vivo. (The latter gratifying results require additional confirmation in other tumor models and with other anticancer agents.)
Chemotherapy has cured a few types of human tumors. However, many human advanced solid cancers respond poorly to chemotherapy because of drug-resistant cells. The ATP-depleting strategy could circumvent the latter problem, and thereby createthe opportunity for chemotherapeutic cure of the more common human malignancies. The belief, and general consensus that chemotherapy kills by apoptosis, and therefore that cells resistant to apoptosis are resistant to drug therapy, neglects thelong-established fact that chemotherapy also kills by necrosis (5-7), and overlooks the evidence that cell death by blocked apoptosis can be "switched" to necrosis (7-9). Overcoming drug resistance is the most important obstacle to the success ofchemotherapy in the cure of advanced human cancers.
REFERENCES
1. King, K. L. and Cidlowski, J. A. Cell cycle and apoptosis: common pathways to life and death. J. Cell Biochem 58: 175-180, 1995. 2. Berns, A. Senescence: A companion in chemotherapy? Cancer Cell: May, 2002; 309-311. 3. Schmitt, C. A.,Fridman, J. S., Yang, M., Lee, S., Baranov, E., Hoffman, R. M., and Lowe, S. W. A senescence program controlled by p53 and p16 ink4a contributed to the outcome of cancer therapy. Cell 109: 335-346, 2002. 4. Schmitt, C. A. and Lowe, S. W. Apoptosis andchemoresistance in transgenic cancer models. J. Mol. Med. 80: 137-146, 2002. 5. Martin, D. S., Bertino, J. R., and Koutcher, J. A. ATP depletion+pyrimidine depletion can markedly enhance cancer therapy: Fresh insight for a new approach. Cancer Res. 60: 6776-6783, 2000. 6. Eguchi, Y., Shimizu, S., and Tsujimoto, Y. Intracellular ATP levels determine cell fate by apoptosis or necrosis. Cancer Res. 57: 1835-1840, 1997. 7. Leist, M., Single, B., Castoldo, A. F., Kuknle, S., and Nicotera, P.Intracellular triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. 185: 1481-1486, 1997. 8. Sane, A. T., and Bertrand, R. Caspase inhibition in camptothecin-treated U-937 cells is completed with ashift from apoptosis to transient G1 arrest followed by necrotic cell death. Cancer Res., 59: 3565-3569, 1999. 9. Lemaire, C., Andreau, K., Souvannavong, K., and Adam, A. Inhibition of caspase activity induced a switch from apoptosis to necrosis. FEBS Lett. 425: 266-270, 1998.
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